For in vitro diagnostic use. Others cobas EGFR Mutation Test v2 IVD IVD cobas® EGFR Mutation Test v2 RMD-4800-EGFRV2-003 07 248 563 190 7 248 563 190 07248563190 7248563190 07248563190 KIT COBAS EGFR AMP/DET V2 24T IVD cobas EGFR Mutation Test v2 00875197005448 Reagents, kits 1 kit 24 tests false The cobas® EGFR Mutation Test v2 is a real-time PCR test for the qualitative detection of defined mutations of the epidermal growth factor receptor (EGFR) gene in non-small cell lung cancer (NSCLC) patients. Defined EGFR mutations are detected using DNA isolated from formalin-fixed paraffin-embedded tumor tissue (FFPET) or circulating-free tumor DNA (cfDNA) from plasma derived from EDTA anti-coagulated peripheral whole blood.The test is indicated as a companion diagnostic to aid in selecting NSCLC patients for treatment with EGFR tyrosine kinase inhibitors in accordance with the approved therapeutic product labeling:Table 1: DrugFFPETPlasmaTARCEVA® (erlotinib)Exon 19 deletions and L858RExon 19 deletions and L858RTAGRISSO® (osimertinib)Exon 19 deletions, L858R and T790MExon 19 deletions, L858R and T790MIRESSA® (gefitinib)Exon 19 deletions and L858RExon 19 deletions and L858RTesting of plasma specimens is most appropriate for consideration in patients from whom a tumor biopsy cannot be obtained. Patients who are negative for these mutations by this test using plasma specimens should be reflexed to routine tissue biopsy and testing for EGFR mutations with the FFPET sample type, if available.Drug safety and efficacy have not been established for the following EGFR mutations also detected by the cobas® EGFR Mutation Test v2:Table 2: DrugFFPETPlasmaTARCEVA® (erlotinib)G719X, Exon 20 insertions, T790M, S768I and L861QG719X, Exon 20 insertions, T790M, S768I and L861QTAGRISSO® (osimertinib)G719X, Exon 20 insertions, S768I, and L861QG719X, Exon 20 insertions, S768I, and L861QIRESSA® (gefitinib)G719X, Exon 20 insertions, T790M, S768I and L861QG719X, Exon 20 insertions, T790M, S768I and L861QThe cobas® EGFR Mutation Test v2 for use with plasma includes a semi-quantitative measurement of mutations in exons 18, 19, 20, and 21 of the EGFR gene. This measurement, reported as a semi-quantitative index (SQI), correlates to the amount of target mutant cfDNA in plasma and can be used to determine changes in target mutant cfDNA load over time for a given patient.For manual sample preparation, FFPET specimens are processed using the cobas® DNA Sample Preparation Kit and plasma specimens are processed using the cobas® cfDNA Sample Preparation Kit. The cobas® z 480 analyzer is used for automated amplification and detection. en The cobas® EGFR Mutation Test v2 is a real-time PCR test for the qualitative detection of defined mutations of the epidermal growth factor receptor (EGFR) gene in non-small cell lung cancer (NSCLC) patients. Defined EGFR mutations are detected using DNA isolated from formalin-fixed paraffin-embedded tumor tissue (FFPET) or circulating-free tumor DNA (cfDNA) from plasma derived from EDTA anti-coagulated peripheral whole blood.The test is indicated as a companion diagnostic to aid in identifying patients with NSCLC for treatment with a tyrosine kinase inhibitor in accordance with the approved therapeutic product labeling:Table 1: DrugFFPETPlasmaEGFR Tyrosine Kinase Inhibitors approved by FDA*Exon 19 Deletions, L858RExon 19 Deletions, L858RAdditional indications in accordance with approved therapeutic product labelingTAGRISSO® (osimertinib)T790MT790M***For the most current information about the therapeutic products in this group, go to:https://www.fda.gov/medicaldevices/productsandmedicalprocedures/invitrodiagnostics/ucm301431.htm.Patients with positive cobas® EGFR Mutation Test v2 test results using plasma specimens for the presence of the EGFR mutations listed above are eligible for treatment with the corresponding drug as indicated in Table 1 (See Note** for T790M). Patients who are negative for these mutations by this test using plasma specimens should be reflexed to routine biopsy and testing for EGFR mutations with the FFPET sample type.Note : **The efficacy of TAGRISSO® (osimertinib) has not been established in the EGFR T790M plasma-positive, tissue-negative or unknown population and clinical data for T790M plasma-positive patients are limited; therefore testing using plasma specimens is most appropriate for consideration in patients from whom a tumor biopsy cannot be obtained.Drug safety and efficacy have not been established for the EGFR mutations listed that are also detected by the cobas® EGFR Mutation Test v2:Table 2: DrugFFPETPlasmaTARCEVA® (erlotinib)G719X, Exon 20 insertions, T790M, S768I and L861QG719X, Exon 20 insertions, T790M, S768I and L861QTAGRISSO® (osimertinib)G719X, Exon 20 insertions, S768I, and L861QG719X, Exon 20 insertions, S768I, and L861QIRESSA® (gefitinib)G719X, Exon 20 insertions, T790M, S768I and L861QG719X, Exon 20 insertions, T790M, S768I and L861QFor manual sample preparation, FFPET specimens are processed using the cobas® DNA Sample Preparation Kit and plasma specimens are processed using the cobas® cfDNA Sample Preparation Kit. The cobas® z 480 analyzer is used for automated amplification and detection. en The cobas EGFR Test is based on two major processes: (1) manual sample preparation to obtain DNA from FFPET or plasma; and (2) PCR amplification and detection of target DNA using complementary primer pairs and oligonucleotide probes labeled with fluorescent dyes. The cobas EGFR Test is designed to detect the following mutations:Exon 18: G719X (G719A, G719C, and G719S)Exon 19: deletions and complex mutations (defined as the combination of a deletion and an insertion)Exon 20: S768I, T790M, and insertionsExon 21: L858R and L861QMutation detection is achieved through PCR analysis with the cobas®z 480 analyzer. A mutant control and negative control are included in each run to confirm the validity of the run.Sample preparationThe cobas® DNA Sample Preparation Kit and the cobas® cfDNA Sample Preparation Kit are for manual sample preparations from FFPET and plasma respectively, based on nucleic acid binding to glass fibers. A protease and chaotropic lysis/binding buffer releases nucleic acids and protects the released DNA from DNases. Subsequently, isopropanol is added to the lysis mixture that is then centrifuged through a column with a glass fiber filter insert. During centrifugation, the DNA is bound to the surface of the glass fiber filter. Unbound substances, such as salts, proteins and other cellular impurities, are removed by centrifugation. The adsorbed nucleic acids are washed and then eluted with an aqueous solution. The target DNA is then amplified and detected on the cobas® z 480 analyzer using the amplification and detection reagents provided in the cobas EGFR Test kitPCR amplificationTarget selectionThe cobas EGFR Test uses primers that define specific base-pair sequences for each of the targeted mutations. For the exon 19 deletion mutations, sequences ranging from 125 to 141 base pairs are targeted; for the L858R substitution mutation in exon 21, a 138 base pair sequence is targeted; for the T790M substitution mutation in exon 20, a 118 base pair sequence is targeted; for the G719X substitution mutation in exon 18, sequences ranging from 104-106 base pairs are targeted; for the S768I substitution mutation in exon 20, a 133 base pair sequence is targeted; for the exon 20 insertion mutations, sequences ranging from 125 to 143 base pairs are targeted; for the L861Q substitution mutation in exon 21, a 129 base pair sequence is targeted. Amplification occurs only in the regions of the EGFR gene between the primers; the entire EGFR gene is not amplified.Target amplificationA derivative of Thermus species Z05-AS1 DNA polymerase is utilized for target amplification. First, the PCR mixture is heated to denature the DNA and expose the primer target sequences. As the mixture cools, the upstream and downstream primers anneal to the target DNA sequences. The Z05 DNA polymerase, in the presence of divalent metal cation and excess dNTP, extends each annealed primer, thus synthesizing a second DNA strand. This completes the first cycle of PCR, yielding a double-stranded DNA copy which includes the targeted base-pair regions of the EGFR gene. This process is repeated for a number of cycles, with each cycle effectively doubling the amount of amplicon DNA.Automated real-time mutation detection The cobas EGFR Test utilizes real-time PCR technology. Each target-specific, oligonucleotide probe in the reaction is labeled with a fluorescent dye that serves as a reporter, and with a quencher molecule that absorbs (quenches) fluorescent emissions from the reporter dye within an intact probe. During each cycle of amplification, the probe complementary to the single-stranded DNA sequence in the amplicon binds and is subsequently cleaved by the 5’ to 3’ nuclease activity of the Z05-AS1 DNA Polymerase. Once the reporter dye is separated from the quencher by this nuclease activity, fluorescence of a characteristic wavelength can be measured when the reporter dye is excited by the appropriate spectrum of light. Four different reporter dyes are used to label the mutations targeted by the test. Amplification of the seven targeted EGFR sequences are detected independently across three reactions by measuring fluorescence at the four characteristic wavelengths in dedicated optical channels.Selective amplification Selective amplification of target nucleic acid from the sample is achieved in the cobas EGFR Test by the use of AmpErase (uracil-N-glycosylase) enzyme and deoxyuridine triphosphate (dUTP). 15 The AmpErase enzyme recognizes and catalyzes the destruction of DNA strands containing deoxyuridine but not DNA containing thymidine. Deoxyuridine is not present in naturally occurring DNA but is always present in amplicon due to the use of dUTP in place of deoxythymidine triphosphate as one of the nucleotide triphosphates in the Master Mix reagents; therefore, only amplicon contains deoxyuridine. Deoxyuridine renders contaminating amplicon susceptible to destruction by AmpErase enzyme prior to amplification of the target DNA. The AmpErase enzyme, which is included in the Master Mix reagents, catalyzes the cleavage of deoxyuridine-containing DNA at the deoxyuridine residues by opening the deoxyribose chain at the C1- position. When heated in the first thermal cycling step at alkaline pH, the amplicon DNA chain breaks at the position of the deoxyuridine, thereby rendering the DNA non-amplifiable. The AmpErase enzyme is inactive at temperatures above 55ºC, i.e., throughout the thermal cycling steps, and therefore does not destroy target amplicon. The cobas EGFR Test has been demonstrated to inactivate deoxyuridine-containing EGFR mutant amplicon.15. Longo M. C., Berninger M. S., Hartley J. L. Use of uracil DNA glycosylase to control carry-over contamination in polymerase chain reactions. Gene. 1990;93(1):125-8. en The cobas EGFR Test is based on two major processes: (1) manual sample preparation to obtain DNA from FFPET or plasma; and (2) PCR amplification and detection of target DNA using complementary primer pairs and oligonucleotide probes labeled with fluorescent dyes. The cobas EGFR Test is designed to detect the following mutations: Exon 18: G719X (G719A, G719C, and G719S) Exon 19: deletions and complex mutations (defined as the combination of a deletion and an insertion) Exon 20: S768I, T790M, and insertions Exon 21: L858R and L861QMutation detection is achieved through PCR analysis with the cobas® z 480 analyzer. A mutant control and negative control are included in each run to confirm the validity of the run.Sample preparationThe cobas® DNA Sample Preparation Kit and the cobas® cfDNA Sample Preparation Kit are for manual sample preparations from FFPET and plasma respectively, based on nucleic acid binding to glass fibers. A protease and chaotropic lysis/binding buffer releases nucleic acids and protects the released DNA from DNases. Subsequently, isopropanol is added to the lysis mixture that is then centrifuged through a column with a glass fiber filter insert. During centrifugation, the DNA is bound to the surface of the glass fiber filter. Unbound substances, such as salts, proteins and other cellular impurities, are removed by centrifugation. The adsorbed nucleic acids are washed and then eluted with an aqueous solution. The target DNA is then amplified and detected on the cobas® z 480 analyzer using the amplification and detection reagents provided in the cobas EGFR Test kitPCR amplificationTarget selectionThe cobas EGFR Test uses primers that define specific base-pair sequences for each of the targeted mutations. For the exon 19 deletion mutations, sequences ranging from 125 to 141 base pairs are targeted; for the L858R substitution mutation in exon 21, a 138 base pair sequence is targeted; for the T790M substitution mutation in exon 20, a 118 base pair sequence is targeted; for the G719X substitution mutation in exon 18, sequences ranging from 104-106 base pairs are targeted; for the S768I substitution mutation in exon 20, a 133 base pair sequence is targeted; for the exon 20 insertion mutations, sequences ranging from 125 to 143 base pairs are targeted; for the L861Q substitution mutation in exon 21, a 129 base pair sequence is targeted; for the internal control in exon 28, a 87 base pair sequence is targeted. Amplification occurs only in the regions of the EGFR gene between the primers; the entire EGFR gene is not amplified.Target amplificationA derivative of Thermus species Z05-AS1 DNA polymerase is utilized for target amplification. First, the PCR mixture is heated to denature the DNA and expose the primer target sequences. As the mixture cools, the upstream and downstream primers anneal to the target DNA sequences. The Z05 DNA polymerase, in the presence of divalent metal cation and excess dNTP, extends each annealed primer, thus synthesizing a second DNA strand. This completes the first cycle of PCR, yielding a double-stranded DNA copy which includes the targeted base-pair regions of the EGFR gene. This process is repeated for a number of cycles, with each cycle effectively doubling the amount of amplicon DNA.Automated real-time mutation detectionThe cobas EGFR Test utilizes real-time PCR technology. Each target-specific, oligonucleotide probe in the reaction is labeled with a fluorescent dye that serves as a reporter, and with a quencher molecule that absorbs (quenches) fluorescent emissions from the reporter dye within an intact probe. During each cycle of amplification, probe complementary to the single-stranded DNA sequence in the amplicon binds and is subsequently cleaved by the 5’ to 3’ nuclease activity of the Z05-AS1 DNA Polymerase. Once the reporter dye is separated from the quencher by this nuclease activity, fluorescence of a characteristic wavelength can be measured when the reporter dye is excited by the appropriate spectrum of light. Four different reporter dyes are used to label the mutations targeted by the test. Amplification of the seven targeted EGFR sequences are detected independently across three reactions by measuring fluorescence at the four characteristic wavelengths in dedicated optical channels.Selective amplificationSelective amplification of target nucleic acid from the sample is achieved in the cobas EGFR Test by the use of AmpErase (uracil-N-glycosylase) enzyme and deoxyuridine triphosphate (dUTP). 15 The AmpErase enzyme recognizes and catalyzes the destruction of DNA strands containing deoxyuridine but not DNA containing thymidine. Deoxyuridine is not present in naturally occurring DNA but is always present in amplicon due to the use of dUTP in place of deoxythymidine triphosphate as one of the nucleotide triphosphates in the Master Mix reagents; therefore, only amplicon contains deoxyuridine. Deoxyuridine renders contaminating amplicon susceptible to destruction by AmpErase enzyme prior to amplification of the target DNA. The AmpErase enzyme, which is included in the Master Mix reagents, catalyzes the cleavage of deoxyuridine-containing DNA at the deoxyuridine residues by opening the deoxyribose chain at the C1- position. When heated in the first thermal cycling step at alkaline pH, the amplicon DNA chain breaks at the position of the deoxyuridine, thereby rendering the DNA non-amplifiable. The AmpErase enzyme is inactive at temperatures above 55°C, i.e., throughout the thermal cycling steps, and therefore does not destroy target amplicon. The cobas EGFR Test has been demonstrated to inactivate deoxyuridine-containing EGFR mutant amplicon.15. Longo MC, Berninger MS, JL Hartley. Use of uracil DNA glycosylase to control carry-over contamination in polymerase chain reactions. Gene. 1990;93:125-128. en

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